Electrospray ionization mass spectrometry (ESI/MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) mass spectrometry, combined capillary electrophoresis (CE) and mass spectrometry, and accurate mass measurement are the techniques of current interest. Preliminary studies of MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix show promise for the rapid analysis of small molecules (molecular weight range = 200-1000). This approach is being compared with conventional MALDI/MS using both normal and unconventional (e.g. graphite) matrices in order to define the advantages and limits of each for small molecule analysis. The off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) and with CE is also under investigation as an alternate approach for the targeted analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for reanalysis. A similar approach is being applied with CE where on-line sample concentration techniques involving electrophoretic focusing of large-volume sample injections prior to peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS. Our goal is to automate the collection process because of the very small (nanoliter) volumes involved. Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) are potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones exhibit varying degrees of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic libraries be rapidly characterized and their structures established before biological evaluation. One method used for the initial characterization of these synthetic DAG-lactone libraries employs a combine-and-analyze strategy with rapid analysis of the resultant mixtures by fast atom bombardment mass spectrometry. This approach correlates directly with individual analysis, substantially reduces the number of samples to be examined, results in enhanced analytical turn-around and is amenable to accurate mass measurement. Neither LC/MS nor flow-injection ESI/MS are suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. However, flow-injection APCI/MS of individual DAG-lactones produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Matrix-less MALDI (see LDI above) is currently under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our goal is the comprehensive structural characterization of all 96 library components in one day. Nucleotide prodrugs of the orally active DNA methyltransferase (DNMT) inhibitor 2(1H)-pyrimidinone riboside (zebularine) continue under investigation. Zebularine exhibits high activity in inhibiting DNA methylation but low potency relative to other known inhibitors of DNMT. It is postulated that incorporation of zebularine into DNA is required before the drug can function as an inhibitor of DNMT by formation of a tight complex between it and the enzyme. We have previously shown that the intracellular metabolic activation of zebularine is both complex and inefficient, resulting in only very limited DNA incorporation. Since DNA incorporation requires the activated 2'-deoxynucleotide, the metabolic bottleneck for DNA incorporation appears to be the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase. 2'-Deoxyzebularine itself shows no inhibitory activity for DNA methylation. We have found that this lack of activity for 2'-deoxyzebularine is due to it being an extremely poor substrate for the mammalian cellular kinases responsible for the initial phosphorylation step. 2'-Deoxyzebularine could be phosphorylated, however, in murine colon cancer cells that had been transfected with a Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK). We observed that 2'-deoxyzebularine possessed a significant antiproliferative effect in these Dm-dNK expressing cells, while it had no cytotoxic effect against wild type tumor cells. Thus, in conjunction with our FDA collaborators, we are making and evaluating thermally and enzymatically activated prodrugs of 2'-deoxzebularine-5'-monophosphate (2'-dZeb-MP) in order to circumvent this metabolically inefficient activation and increase drug potency. We have demonstrated that this prodrug concept is feasible in principle by showing that 2'-dZeb-MP can be generated from a cyclic(2'-dZeb-MP) dimer with thermally labile protecting groups under physiological conditions. Analysis by CE and MS showed that removal of the phosphate protecting groups occurs sequentially with similar rate constants and that the resulting dinucleotide dimer can be converted to 2'-dZeb-MP by phosphodiesterase-I. Our immediate goal is to correlate the kinetics of activation of various cyclic(2'-dZeb-MP) dimer derivatives with their in vitro activity